The goals and aims of the grant are based on previous studies showing that Qa-1 restricted, TCR Vbeta specific CD8+ T cells play a major role in regulating peripheral self-reactive TCR repertoire by selectively down-regulating potentially pathogenic self-reactive T cells. Our observations lead us to propose an "affinity model" of peripheral T cell regulation by the regulatory CD8+ T cells. Basically, the affinity model proposes that whether or not a CD4+ T cell will express Qa-1/self-peptide on their surface to induce and be specifically regulated by CD8+ T cells is dependent on the affinity of their TCR interaction with antigen/MHC class II complex on APCs during the initial antigen triggering. In this regard, it is known that following T cell activation the diversity of peptides bound to Qa-1 increases so that the activated T cells express Qa-1 molecules not only bound to the canonical Qa-1 binding peptide, Qdm, but also to self-peptides induced by T cell activation. We hypothesize that these new Qa-1/self-peptide complexes are recognized by regulatory CD8+ T cells which then differentiate to down-regulate activated CD4+ T cells expressing these novel Qa-1/self-peptide. In this grant we intend to test the "affinity model" by studying the immune responses to 1-9Nac MBP in mono-phase EAE model. The affinity/avidity of MBP reactive CD4+ T cells will be measured by antigen dose response curve methods (ED50) as well as by MHC class II tetramer binding. Qa-1 binding peptide/s in EAE will be identified and tested in collaboration with Dr. Mark Soloski. We will also extend our studies of regulatory CD8+ T cells in control of auto-immunity to a chronic EAE model induced by MOG 33-55 in B6 mice (Qa-1b strain) which more closely mimics human MS. This will not only allow us to focus on more clinically oriented studies for further applications of the concepts of Qa-1 restricted regulatory CD8+ T cells, but also permit direct studies of both in vivo and in vitro detail molecular and cellular mechanisms of Qa-1 restriction in this T-T cell interaction. The peripheral MOG 33-55 reactive repertoire in B6 mice will be analyzed by TCR Vbeta usage, RT-PCR based CDR3 length distribution analysis and CDR3 sequencing. In addition, mechanisms of a potential therapeutic agent (anti-CD3) ameliorating EAE will be studied. The overall goal of this grant is to understand not only the theoretical basis of the immuno-regulatory pathway mediated by the Qa-1 restricted CD8+ T cells in control of auto-immunity but also the potential application of this pathway in treating auto-immune disease. In this regard, our studies will provide new insight into the general biology of peripheral T cell regulation and basic mechanisms by which the immune system could be manipulated to specifically suppress pathogenic auto-immune responses.